**Aflatoxin Contamination and Research in China**

Huili Zhang1,2, Jianwei He2, Bing Li3,

Hui Xiong3, Wenjie Xu3 and Xianjun Meng1 *1Academy of Food Science Shenyang Agricultural University, Shenyang, 2School of Life Science, Liaoning University, Shenyang, 3College of Light Industry, Liaoning University, Shenyang, China* 

#### **1. Introduction**

20 Aflatoxins – Detection, Measurement and Control

United States Department of Agriculture Foreign Agricultural Service (2011) Cheese

United States Department of Agriculture Foreign Agricultural Service. Cheese Production

Van Egmond, H.P. (1989) Aflatoxin M1: Occurrence, toxicity, regulation. In *Mycotoxins in* 

Van Egmond, H.P.; Dragacci, S .(2001) Liquid Chromatographic Method for Aflatoxin M1 in

Virdis, S.; Corgiolu, G.; Scarano, C.; Pilo, A.L.; De Santis, E.P.L. (2008) Occurrence of

Wang, J. J.; Liu, B.H.; Hsu, Y.T.; Yu, F.Y (2011) Sensitive competitive direct enzyme-linked

Yousef, A. E.; Marth, E. H. (1989) Stability and degradation of aflatoxin M1. In: *Mycotoxins in* 

and Consumption: Summary For Selected Countries 21/04/2011. Available from: http://www.fas.usda.gov/psdonline/psdReport.aspx?hidReportRetrievalName= Cheese+Production+and+Consumption%3a+Summary+For+Selected+Countries&

*Dairy products*. Van Egmond H.P. (Ed.) 11-59 Elsevier Applied Science Publisher,

Milk. In: *Methods in Molecular Biology, Vol 157 Mycotoxin Protocols II*. M.W. Trucksess; A.E. Pohland (Ed) 59-69 Humana Press, ISBN 0-89603-623-5, Totowa

Aflatoxin M1 in tank bulk goat milk and ripened goat cheese. *Food Control* Vol. 19,

immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk *Food Control* Vol. 22, pp. 964-969, ISSN 0956-7135 Yapar, K.; Elmali, M.; Kart, A.; Yaman, H. (2008) Aflatoxin M1 levels in different type of

cheese products produced in Turkey *Medycyna Weterynaryina* Vol. 64, No. 1, pp. 53-

*dairy products*. Egmond, H. P. van (Ed) 127-161 Elsevier Applied Science Publisher,

Production Selected Countries. 21/04/2011. Available from: (http://www.fas.usda.gov/dlp2/circular/1998/98-01Dairy/cheese.pdf)

hidReportRetrievalID=1233&hidReportRetrievalTemplateID=7

ISBN 1-85166-369-X, London, United Kingdom

ISBN 1-85166-369-X, London, United Kingdom

(NJ), United States

55 ISSN 00258628

pp. 44–49, ISSN 0956-7135

Aflatoxins(AF) are highly poisonous secondary metabolites produced by *Aspergillus flavus* and *Aspergillus parasiticus*. They have been found in moldy human food and animal feeds and have been implicated in numerous animal disorders. *A. parasiticus* produces four major aflatoxins: B1 , B2 , G1 and G2 , while AFB1 is the most toxic in the group and the toxicity is in the order of B1 > G1 > B2 > G2. Since the 1960 outbreak of Turkey X disease, when more than 10,000 turkeys died after being fed with aflatoxin contaminated peanut meal, scientists in China have paid more attention to the studies on aflatoxins including its distribution, pollution, health hazards, testing, monitoring, detection technology, managing, microbiology, ecology, toxicology, and policies in controlling aflatoxins. In this review, we present a brief report on the situation of aflatoxin contamination and research progress in China.

#### **2. Distribution of aflatoxin contamination**

#### **2.1 The distribution in cereals, oils and foodstuffs**

In general, the nationwide aflatoxin contamination was mainly in cereals, oils and foodstuffs. The aflatoxin B1 (AFB1) content detected in vegetable oil products was far higher than in food products. Based on 1,000 investigations of susceptible aflatoxin contamination from nearly 20 provinces between 2002 and 2008, contamination was reported in almost every province. The majority of the samples tested shows the presence of aflatoxins. The overall level of contamination in southern part of China is higher than in the northern region. The most severe province is Guangxi. The main reason is due to the hot and humid southern climate. Climatic condition significant influences the level of aflatoxin contamination. When in serious drought and/or high temperature conditions, or when the soil humidity is below the normal level before harvest, it increase in the number of *A. flavus* spores in the air resulting more fungal infection, and thus high level aflatoxin accumulation. During end processing and packaging, storage of animal-derived food, "cold chain" transfer or pollution of the packing material could also lead to the *A. flavus* infection and aflatoxin contamination (Duan *et al.*, 2009).

Aflatoxin Contamination and Research in China 23

mycoprotein, respectively (Wang *et al.*, 2003). The data showed the relevant ratio, average content and above limit ratio of aflatoxins in feedstuffs were 92.1%, 8.15g/kg and 6.6%, respectively. These values were 100%and 5.95μg/kg in dairy cattle mix feed as studied

The investigation demonstrated that the safety of fermented flavoring food products such as soy sauce is very optimistic in China. A latest survey of 203 samples of national brand soy sauce samples in 2010 showed that the aflatoxin level is below the maximum allowed level set forth by European Commission (Qi & Che, 2010). This may be contributed by the fact that soybean,row material of fermentation, is not susceptible to infection of aflatoxinproducing fungi preharvest, eventhough the growth condition of *Aspergillus oryzae* and

The maximum amount AFB1 allowed in brewed soy sauce in China was set by law at 5μg/kg. In order to understand the AFB1 contamination of the brewed soy sauce in China, 203 soy sauce samples from different provinces in China were tested for the establishment of emergency response and early warning systems of AFB1 (Sun *et al.*, 2010). The study concluded that the soy sauce is safe for consumption. The average AFB1 content in the brewed soy sauce from the five provinces in China were 0.3560μg/kg, 0.4636μg/kg, 0.5273μg/kg, 0.3143μg/kg and 0.2083μg/kg respectively. All of the tested samples were bellow the maximum allowed level set forth in China and the EU countries,

Due to a great variety of traditional Chinese medicines and the wide area of planting regions, the traditional Chinese medicinal herbs can be infected with aflatoxin-producing fungus, *A. flavus*, in the process of processing, storage and transportation. Aflatoxin-producing fungus exists in soil and air and Chinese medicinal herbs can be infected by *A. flavus* directly. Studies demonstrated previously that the aflatoxin contamination in Chinese herbal medicine is

The investigation of AFB1 in regular Chinese herbal medicine and Chinese traditional patent medicines using the method of ELISA has been reported (Ren & Ma, 1997). It was demonstrated that the presence of AFB1 in traditional Chinese medicinal materials was common. Results suggested that the positive rate and contents of AFB1 were serious enough to alert our concern. Studies in 20 different provinces during 1997~2001 period showed that the AFB1 content in 83%~100% samples was over the limits allowed, with several samples seriously over limits. The indirectly competitive enzyme-linked immunosorbent assay on seven Chinese medicines showed that the aflatoxin content in severe cases reached as high as 200~229 ng/g in Shenqu and 1,056 ng/g in Yueju baohe pellet, respectively (Liu, 2001). Other report showed 85% of samples detected the presence of aflatoxin at a concentration

A series following surveys of nearly 10,000 people from 2006 to 2009 show that the toxicity has a positive correlation with the distribution of AFB1. The total morbidity in the southern region is more serious than in the northern region. Guangxi and Zhejiang

*Aspergillus niger*, are similar to that of the aflatoxin-producing fungi.

another issue of concern in preventing aflatoxin contamination in the food.

**2.5 The distribution in traditional chinese medicine** 

during 2006 and 2007 (Ao & Chen, 2008).

**2.4 Distribution in fermented flavoring** 

which is 2μg/kg.

less than 1ng/g (Tang, 1999b).

**3. The toxicity of aflatoxin** 

Studies on the level of aflatoxin B1 in 486 foodstuff and 146 oil samples collected from 18 cities in 2008 (Zhang, 2008) demonstrated that the levels of aflatoxin contamination were between 0.02 and 54.20 μg/kg in foodstuff and 0.41 and 36.54 μg/kg in oil products respectively. While the detection rate ranged from 0.41% to 2.06%, respectively. A similar study in 2004 reported that the aflatoxin B1 detection rate was as high as 58% in 17 grain samples, which was the most severe incidence in Guangxi province (Wang, 2004). Samples collected from 5 provinces including Sichuan (mainly in Chongqing), Guangdong, Guangxi, Hubei and Zhejiang showed that the aflatoxin B1 detection rates were 70.27% and 24.24% in corn and peanut respectively (Liu, 2006). The aflatoxin B1 detection rates in peanut oil, peanut and corn samples collected from Yunnan province were 100%, 24.32% and 5.26% respectively. The aflatoxin levels in samples were 16.05% above the legally allowed limit, which was similar to the level in peanut samples from Beijing (Wei, 2002; Gao et al., 2007) .

#### **2.2 Distribution in dairy products**

The investigation of aflatoxin contamination in dairy products indicated that aflatoxin M1 (AFM1), a hydroxylated metabolite of AFB1 secreted in milk, was commonly detectable in most of the dairy samples tested. This phenomenon is correlated well with the distribution of AFB1. From a survey of more than 1,000 samples in 17 provinces between 1991 and 2005, the following reported detection rate ranged from 4.0% to 73.7%.

The scientists of Guangxi Anti-Epidemic and Basic Course Section monitored the AFM1 contamination in 100 samples of milk and dairy products in 15 provinces between 1991 and 1999,the AFM1 contamination levels in milk were from 0.2 μg/kg to 1.9 μg/kg (Tang, 1999a). In 1991, a study using the HPLC on 57 milk samples and 15 milk powder samples from Shanghai,the detection rate of AFM1 were 26.7% and 73.7%, respectively (concentration ranges between 0.025 and 0.95 μg/kg (Zhu, 1991). A similar survey was performed using TLC in 1995 on 59 milk and 53 milk powder samples from Fuzhou,reported that the detection rates were 4.0% and 13.19% respectively (concentration range between 0.06 and 0.20g/kg (Lin, 1995).

The data indicated that the detection ratio of AFM1 correlate with the high content of AFB1 in animal feed. The amount of AFB1 consumption by animals influences the amount of AFM1 secreted in milk in a dose-dependent manner. Again, the highest level was detected in milk and dairy products from Guangxi province.

#### **2.3 Distribution in feed**

The investigation of aflatoxin contamination in animal feed demonstrated the wide distribution of aflatoxins. A survey done in more than 1,000 samples in 20 provinces from 2003 to 2008 showed that aflatoxins present in most of the samples, as stated below. This analysis indicated that the general level of serious contamination in southern region is similar to that in northern region. Irradiation has been suggested as a possible means of controlling insects and microbial populations in stored food under moist storage condition (Xiao *et al.*, 2007).

Aflatoxins in feeds has long been a problem in Huanan, Huabei and Huazhong large geographic regions. Detections of AFB1 in 109 samples showed that the aflatoxin detectable rate and the average content were 83.9% and 24.6μg/kg in corn, 100% and 8.27μg/kg in complete feed, 100% and 6.81μg/kg in animal and plant protein, 100% and 13.3μg/kg in

Studies on the level of aflatoxin B1 in 486 foodstuff and 146 oil samples collected from 18 cities in 2008 (Zhang, 2008) demonstrated that the levels of aflatoxin contamination were between 0.02 and 54.20 μg/kg in foodstuff and 0.41 and 36.54 μg/kg in oil products respectively. While the detection rate ranged from 0.41% to 2.06%, respectively. A similar study in 2004 reported that the aflatoxin B1 detection rate was as high as 58% in 17 grain samples, which was the most severe incidence in Guangxi province (Wang, 2004). Samples collected from 5 provinces including Sichuan (mainly in Chongqing), Guangdong, Guangxi, Hubei and Zhejiang showed that the aflatoxin B1 detection rates were 70.27% and 24.24% in corn and peanut respectively (Liu, 2006). The aflatoxin B1 detection rates in peanut oil, peanut and corn samples collected from Yunnan province were 100%, 24.32% and 5.26% respectively. The aflatoxin levels in samples were 16.05% above the legally allowed limit, which was similar to the level in peanut samples from

The investigation of aflatoxin contamination in dairy products indicated that aflatoxin M1 (AFM1), a hydroxylated metabolite of AFB1 secreted in milk, was commonly detectable in most of the dairy samples tested. This phenomenon is correlated well with the distribution of AFB1. From a survey of more than 1,000 samples in 17 provinces between 1991 and 2005,

The scientists of Guangxi Anti-Epidemic and Basic Course Section monitored the AFM1 contamination in 100 samples of milk and dairy products in 15 provinces between 1991 and 1999,the AFM1 contamination levels in milk were from 0.2 μg/kg to 1.9 μg/kg (Tang, 1999a). In 1991, a study using the HPLC on 57 milk samples and 15 milk powder samples from Shanghai,the detection rate of AFM1 were 26.7% and 73.7%, respectively (concentration ranges between 0.025 and 0.95 μg/kg (Zhu, 1991). A similar survey was performed using TLC in 1995 on 59 milk and 53 milk powder samples from Fuzhou,reported that the detection rates were 4.0% and 13.19% respectively

The data indicated that the detection ratio of AFM1 correlate with the high content of AFB1 in animal feed. The amount of AFB1 consumption by animals influences the amount of AFM1 secreted in milk in a dose-dependent manner. Again, the highest level was detected

The investigation of aflatoxin contamination in animal feed demonstrated the wide distribution of aflatoxins. A survey done in more than 1,000 samples in 20 provinces from 2003 to 2008 showed that aflatoxins present in most of the samples, as stated below. This analysis indicated that the general level of serious contamination in southern region is similar to that in northern region. Irradiation has been suggested as a possible means of controlling insects and microbial populations in stored food under moist storage

Aflatoxins in feeds has long been a problem in Huanan, Huabei and Huazhong large geographic regions. Detections of AFB1 in 109 samples showed that the aflatoxin detectable rate and the average content were 83.9% and 24.6μg/kg in corn, 100% and 8.27μg/kg in complete feed, 100% and 6.81μg/kg in animal and plant protein, 100% and 13.3μg/kg in

the following reported detection rate ranged from 4.0% to 73.7%.

(concentration range between 0.06 and 0.20g/kg (Lin, 1995).

in milk and dairy products from Guangxi province.

**2.3 Distribution in feed** 

condition (Xiao *et al.*, 2007).

Beijing (Wei, 2002; Gao et al., 2007) .

**2.2 Distribution in dairy products** 

mycoprotein, respectively (Wang *et al.*, 2003). The data showed the relevant ratio, average content and above limit ratio of aflatoxins in feedstuffs were 92.1%, 8.15g/kg and 6.6%, respectively. These values were 100%and 5.95μg/kg in dairy cattle mix feed as studied during 2006 and 2007 (Ao & Chen, 2008).

#### **2.4 Distribution in fermented flavoring**

The investigation demonstrated that the safety of fermented flavoring food products such as soy sauce is very optimistic in China. A latest survey of 203 samples of national brand soy sauce samples in 2010 showed that the aflatoxin level is below the maximum allowed level set forth by European Commission (Qi & Che, 2010). This may be contributed by the fact that soybean,row material of fermentation, is not susceptible to infection of aflatoxinproducing fungi preharvest, eventhough the growth condition of *Aspergillus oryzae* and *Aspergillus niger*, are similar to that of the aflatoxin-producing fungi.

The maximum amount AFB1 allowed in brewed soy sauce in China was set by law at 5μg/kg. In order to understand the AFB1 contamination of the brewed soy sauce in China, 203 soy sauce samples from different provinces in China were tested for the establishment of emergency response and early warning systems of AFB1 (Sun *et al.*, 2010). The study concluded that the soy sauce is safe for consumption. The average AFB1 content in the brewed soy sauce from the five provinces in China were 0.3560μg/kg, 0.4636μg/kg, 0.5273μg/kg, 0.3143μg/kg and 0.2083μg/kg respectively. All of the tested samples were bellow the maximum allowed level set forth in China and the EU countries, which is 2μg/kg.

#### **2.5 The distribution in traditional chinese medicine**

Due to a great variety of traditional Chinese medicines and the wide area of planting regions, the traditional Chinese medicinal herbs can be infected with aflatoxin-producing fungus, *A. flavus*, in the process of processing, storage and transportation. Aflatoxin-producing fungus exists in soil and air and Chinese medicinal herbs can be infected by *A. flavus* directly. Studies demonstrated previously that the aflatoxin contamination in Chinese herbal medicine is another issue of concern in preventing aflatoxin contamination in the food.

The investigation of AFB1 in regular Chinese herbal medicine and Chinese traditional patent medicines using the method of ELISA has been reported (Ren & Ma, 1997). It was demonstrated that the presence of AFB1 in traditional Chinese medicinal materials was common. Results suggested that the positive rate and contents of AFB1 were serious enough to alert our concern. Studies in 20 different provinces during 1997~2001 period showed that the AFB1 content in 83%~100% samples was over the limits allowed, with several samples seriously over limits. The indirectly competitive enzyme-linked immunosorbent assay on seven Chinese medicines showed that the aflatoxin content in severe cases reached as high as 200~229 ng/g in Shenqu and 1,056 ng/g in Yueju baohe pellet, respectively (Liu, 2001). Other report showed 85% of samples detected the presence of aflatoxin at a concentration less than 1ng/g (Tang, 1999b).

#### **3. The toxicity of aflatoxin**

A series following surveys of nearly 10,000 people from 2006 to 2009 show that the toxicity has a positive correlation with the distribution of AFB1. The total morbidity in the southern region is more serious than in the northern region. Guangxi and Zhejiang

Aflatoxin Contamination and Research in China 25

In 1970~1999, there were 4,215 new liver cancer cases in Zhongshan. Its crude incidence rate, China and world standardized rates were 13.0/105, 12.5/105, 16.8/105,respectively. There is no increasing or decreasing trend for its incidence rates in 1975~1994. However, a declining tendency between 1995 and 1999 was observed. The liver cancer incidence rate during this period in Zhongshan was moderate comparing with the worldwide statistics, but at middle-high level and at low level compared with urban and rural pilot areas in

The crude and standardized incidences of liver cancer were analyzed by collecting the disease information from the rural area in Ningbo from 2006 to 2008 (Cui *et al.*, 2009). The results show that the crude incidence of hepatoma of the rural residents in Ningbo from 2006 to 2008 is 38.66/105. The age standardized incidence of this disease is 32.14/105. The incidence of hepatoma increased with age. Its incidence in male is 2.77 times of that in female. As to the diagnosis technology, imageology is the most persuasive method to make a definite diagnosis with a ratio of 58.93%. Next effective method is the pathological examination with a ratio of 36.72%. Hepatoma incidence of rural residents in Ningbo is

Aflatoxin exposures can begin in utero and continue through childhood. A mutation in the P53 tumor suppressor gene from AGG to AGT (arginine to serine) transversion at codon 249 (Ser249 mutation) has been reported for hepatocellular carcinoma and matched plasma DNA found in plasma of young children from a region of high aflatoxin exposure (Xu, 2009). This gene mutation in tumor-derived DNA has recently been detected in plasma or serum DNA from adult hepatocellular carcinoma patients. The presence of this mutation before hepatocellular carcinoma onset (e.g., in patients with cirrhosis and patients without clinically diagnosed liver disease) may indicate that the mutation is a marker of chronic exposure to aflatoxin (Kirk *et al.*, 2005). This mutation has been detected in areas with high aflatoxin exposure while it is rare in the low aflatoxin exposure regions

A close relationship between the expression of survivin, a newly founded inhibitor of apoptosis protein (IAP), and the abnormality of Wnt signal transduction pathway, was revealed (Ban & Cao, 2005), (Jiao *et al.*, 2007). The HBV is prevalent in high hepatic cancer risk areas, so there is a synergetic effect between the two risk factors. Using populationbased case-control study to find the main risk factors of hepatocellular carcinoma (HCC), when people exposed with three main environmental factors (HBsAg, intake of moldy food and drinking raw water), the ORs of hepatic cancer were increased by several times

Concerning the coordinate cancergenic mechanism between AFB1 and HBV, it is concluded that: i. both of the risk factors can reduce the gene expression level of drug-metabolizing enzyme; ii. Chronic inflammatory reaction increased possibility of p53 mutation induced by AFTB1. iii. Chronic infection of HBV changes AFTB1 to an active form. iv. HBX inhibits the nucleus excision repair of DNA, hindering the repair of AFB1-DNA adduct and similar DNA damage and accelerating the process of carcinomatous change of hepatic cells. Besides, the sensibility of host to AFB1 and fatty degeneration of liver could also come to be

above the average ratio of that in Zhejiang province and China.

The aflactoxin can result in cancer by a variety of molecular mechanisms.

suggesting a conjugated effect between HbsAg and AFB1 albumin adduct.

China at the same time period.

**3.1.1.2 Pathogenesis** 

(Duan *et al.*, 2005).

carcinogens (Xu, 2009).

provinces are the high incidence regions, which is significantly higher than the already reported AFB1 contamination area, Guangdong, Hunan and Singapore. It is widely accepted that aflatoxin contamination in the region is correlated well with the onset of liver cancer in human. Studies showed that AFB1 causes the p53 gene mutation in human cancer cell. P53 is a tumor suppressor, a transcription factor involved in the regulation of the cell cycle.
